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The purpose of this study was to clarify the mechanism responsible for the transcriptional regulation of the mouse Col5a3 gene in osteoblastic cells. Transient transfection into rat osteosarcoma ROS17/2.8 cells demonstrated that a region from nucleotides 337 to 1 was involved in the transcriptional activity of the Col5a3 gene. An electrophoretic mobility shift assay showed that Sp1/Sp3 and CBF/NF-Y bound to a GC-rich domain (194/186) and a CCAAT box (134/130) in the Col5a3 gene, respectively. Introduction of mutations or deletion into a GC-rich domain, the CCAAT box, or both elements decreased the transcription activity. Overexpression of Sp1 increases the transcription activity and interferes with Sp family binding to the GC-rich domain to decrease promoter activity. Therefore, the transcription of the mouse Col5a3 gene is cooperatively regulated by Sp1 and CBF/NF-Y in osteoblastic cells.

The purpose of this study was to search for chromosomal susceptibility loci for comitant strabismus. Genomic DNA was isolated from 10mL blood taken from each member of 30 nuclear families in which 2 or more siblings are affected by either esotropia or exotropia. A genome-wide search was performed with amplification by polymerase chain reaction of 400 markers in microsatellite regions with approximately 10 cM resolution. For each locus, non-parametric affected sib-pair analysis and non-parametric linkage analysis for multiple pedigrees (Genehunter software, http://linkage.rockefeller.edu/soft/) were used to calculate multipoint lod scores and non-parametric linkage (NPL) scores, respectively. In sib-pair analysis, lod scores showed basically flat lines with several peaks of 0.25 on all chromosomes. In non-parametric linkage analysis for multiple pedigrees, NPL scores showed one peak as high as 1.34 on chromosomes 1, 2, 4, 7, 10, 15, and 16, while 2 such peaks were found on chromosomes 3, 9, 11, 12, 18, and 20. Non-parametric linkage analysis for multiple pedigrees of 30 families with comitant strabismus suggested a number of chromosomal susceptibility loci. Our ongoing study involving a larger number of families will refine the accuracy of statistical analysis to pinpoint susceptibility loci for comitant strabismus.</P>

For the purpose of forecasting the prevalence ofJapanese encephalitis in Japan, we tried to find out the correlation of factors between median and mode dates of epidemic time curve of prevalence on one hand, and average atmospheric temperatures of prefectures in June and July (T6,7 in short) (X¹), the time when HI reaction of swine became positive to the degree of 50 per cent (D. pos. swine in short) (X²), the latitude (x³) and longitude (x4) in respective prefectures (in 1965 and 1967). On the other we also estimated the median and mode dates of this epidemic curve of the prevalence in 1968 and 1969, from the regression equation of one variable and multiple regression equation from the above factors using an electronic computer. The usefulness of adding factors concerned with mosquitoes to the above four factors is proven by the accuracy of estimation. And the following results were obtained. 1) Phenomenally speaking, the prevalence of Japanese encephalitis follows the principle of "advancing of prevalence towards the north and east" and essentially speaking, it depends upon high atmospheric temperature and the outbreak of many hazardous mosquitoes by the high atmospheric temperature. 2) To estimate median date (y) and mode rate (z) of the epidemic time curve of the prevalence, we can use the next equations; The regression equations to estimate y and z from T 6,7(X) are as follows. y = - 3. 75X¹ + 144.47 σ = 12.4.·. [1] z = - 3. 80X¹ + 157 .26 σ = 14.9.. · [1]' The regression equation from D. pos. swine (X²) are as follows. y = 0. 68X² + 31. 82 σ = 9.2· .. [2] z=0. 76X² +40. 71 σ= 12.0 .. · [2]' The multiple regression equation from T6 ,7 and D. pos. swme are as follows. y = -1. 07X¹ +0 .62x² +59. 37 σ= 9.7 ... [3] z= -0. 79x¹ +0. 71x² +61.02 σ= 12.0· .. [3]' The multiple regression equations from T 6•7, D. pos. swine, latitude and longitude are as follows Y= -1.01x¹ +0.58x² -0.26x³+0 .37x4 + 18.50 σ= 9.8･･･ [4] z = -0. 32x¹ +0. 52x² +2 .05x³ +0 .54x4 -87. 81 σ= 11.8 [4]' 3) We Obtained the estimated value of median date in 17 prefectures in Kyushu, Chugoku, Shikoku, Kinki and Kanto provinces in 1968 and in 13 prefectures in 1969 from [l] or [2] or [3] or [4] equation. Nine prefectures out of 17 by [l], 12 prefectures by [2], 13 by [3J and [4] in 1968. [4] could be estimated with about 10 days error or less. And in 1969, 9 out of 13 by [3] and 7 out of 13 by [4] could be accurately esti· mated. The estimation by the multiple regression equation using many factors is most useful for the calculation. 4) The time when the number of patients increases at maximum can be pointed out by the lower limit of prediction region obtained from data in each prefecture. And the lower limit was the estimated median value minus about 20 days by [1] and about 16 days by [2] or [3] or [4] under the next condition; α = 0. 1, N= 75. 5) The mode dates in 17 prefectures out of 19 were estimated by [1]', [2]', [3]' and [4]'. 12 prefectures out of 17 by [1]', 7 by [2]', 10 by [3]' and 13 by [4]' could be estimated with about 12 days error or less in 1968 and 9 out of 13 was correctly estimated by [3]' and [4]' in 1969. The estimation by the regression line of one factor was s~mewhat different from each other, but when multiple regression line of four factors was used the estimation became more correct. Judging from these results, it is adequate to use the multiple regression equation of [4] and [4]' when we want to forecast the median date or mode date ofJapanese encephalitis time cure. 6) In the case of adding two factors concerned with mosquitoes to T6,7 (X¹), D. pos. swine (x²), latitude (x³), longitude (x4), multiple regression equations become as follows. y= -1.46x¹+0.14X²+0.068x5+89.03 σ= 6.9.. ·[5] z= -3. 29x¹+0 .13x²-0. 010x5+ 143.63 σ= 18.6··· [5]' y=-4.20x¹+0.35x²+0.29x6 + 53.70 σ= 4.2 .. ·[6] z=-2.56x¹-0.0lx²-0.02x6 +128.96 σ=11.4 [6]' y= 4.76x¹+0.41x²+0.13x5+0.22x6-72.78 σ= 4.5 [7] z = - 2. l0x¹ + 0. 05x²+ 0. 11 x5 - 0. 08x6+ 113.4 σ= 10. 7.. · [7]' where x5 is the time when the number of mosquitoes (C. T. collected by light trap reached the maximum and X6 is the time when hazardous mosq uitoes were dected. In the case of median date, 5 prefectures out of 6 prefectures by [5], 2 out of 6 by [6] and 2 out of 5 by [7], and in the case of mode date, 5 out of 6 by [5]', 4 out of 5 by [6]' and 4 out of 5 by [7]' could be accurately estimated in 1969.

Antigenicity of the peptide of ribosomal digest in Ehrlich ascites tumor was studied. The peptide was purified by DEAE Sephadex A-50 column chromatography. The peptide was electrophoretically basic, single, and 1.32 S20w sedimentation coefficient with poor content of tyrosine and phenylalanine. The maximum absorbancy was at 267 mμ. Mice and rabbits were immunized with the mixture of the purified peptide with Freund's complete adjuvant. The inhibitory effect of immune γ-globulin on the tumor growth was demonstrated in vitro cultured JTC-11 cells. A single precipitin line was observed between rabbit antiserum and tumor cell extract of Ehrlich ascites cells in ouchterlony double diffusion chamber and immunoelectrophoresis. The sedimentation coefficient of the effective fraction in immune-serum was 17 S20w. The precipitin line was observed at β2-γ region in immunoelectrophoresis.

Exfoliative cytologic studies on normal oral cavity and on the postexodontic wound healing of infants were carried out and the following results were obtained: 1) The keratinization of cells was found marked in such regions as buccal mucosa, mucobuccal fold, gingiva and palate in that order. 2) As for changes in the distribution of cells and leucocytes, the cell distribution in the period of 15-21 postexodontic days proved to be identical with that of normal exfoliated cells. Namely, the wound healing from exfoliative cytologic aspects takes place during the period of 15-21 postexodontic days.

Epidemiological study on the prevalence of SMON III Ibara City, Yoshii Town and Yubara Town in Okayama Prefecture was conducted and the following results were obtained. "Epidemization precession" (Die Prazession der Durchseuchung), described by Rudder which shows that ages common to this disease is shifting to younger generation as incidence rate increases, in the endemic regions was recognized in Ibara City and Yubara Town. The mode of the distribution of the interval between the primary and secondary patients in a family suggests that it corresponds to the incubation period. Distribution of the patients to the size of family is not adaptable to binomial model, but adaptable to chain binomial model, and the intra-household transmitted rate is 5 per cent, and the true intra-household transmitted rate is about two times as high as extra-household transmitted rate by the modified chain binomial model. Intra-hospital incidence among SMON patients and non-SMON patients as well as medical workers were observed at the time when the number of new patients increased in a hospital. In SMON patients, the rate of appendectomized ones was higher than that of non-SMON patients, suggesting that gastrointestinal tract has some relationship to the development of SMON disease.

As a step in the elucidation of immunity of human cancer from the standpoint of homotransplantation immunity, we conducted mixed cultures of regional lymph node cells from C3H mouse isotransplanted with methylcholanthrene-induced sarcoma (MC-tumor) together with the primary culture MC-tumor cells, and observed the behaviors of these lymph node cells to the MC-tumor cells and compared the effects of these lymph node cells with lhose of normal mouse lymph node cells by counting the growth number of tumor cells and also by cinematography. As a result, it has been demonstrated that the regional lymph node cells from the mouse isotransplanted with the MC-tumor (2mm3 in size) acquire a strong antitumor activity by 14 days after the transplantation, but such antitumor activity diminishes and disappears in the terminal stage of cancer. When the number of these lymphocytes is increased, there can be observed some dosage effect, but no complete inhibition of the tumor growth can be attained. The cinematographic observations of these regional lymph node cells in the mixed culture with tumor cells reveal that lymphocytes of small to intermediate size aggregate onto the tumor cells and inhibit the movement of the latter.

1) Three different types of muscle fibers were clearly distinguished in the intercostal muscles and the diaphragm of human, cat and rat by histochemical demonstration of oxidative enzymes, phosphorylase and glycogen. 2) The intercostal muscles and diaphragm each presented dissimilar patterns of the muscle fibers. The diaphragm did not show any definite correlation between the diameter and the histochemical reaction of muscle fibers but its red fibers indicated a more intense uptake of Nitro-BT formazan deposits in the subsarcolemmal region. In this conection, the relationship between the motor innervation and histochemical evaluation of the diaphragm was described. 3) Phosphorylase and glycogen showed reciprocal reactions to the oxidative enzyme activity. They were generally high in large fibers but low in small fibers, and moderate in intermediate fibers.

For purpose to study specificity in the growth inhibition effect of sensitized lymph-node cells on target cells, the regional lymph-node cells obtained from the truly isologous mouse previously inoculated with A strain cells (derived from C3H mouse mammary cancer) were cultured with A cells in various ways, and obtained the following results. 1. Those regional lymph-node cells from the isologous mice transplanted with the skin of C3H mouse or MC-induced sarcoma do not inhibit the growth of A cells in tissue culture. 2. The regional lymph-node cells from the mice positive to tuberculin test also do not inhibit the growth of A cells in tissue culture. 3. The regional axillary lymph-node cells (C3H anti-A strain cells) inhibit the proliferation of M cells from Cb mouse mammasy cancer and JTC-ll cells from Ehrlich ascites tumor as well as A cells. However, these axillary lymphnode cells do not inhibit the growth of AH-66F cells from rat DAB hepatoma, Hela-S3 cells from human uterine cancer and L cells from subcutaneous connective tissue of C3H mouse. From these results it is assumed that the sensitized regional lymph- node cells act specifically on cancer antigen.

As a link in the series of studies on tumor-specific immunity, in vitro inhibitory effect of sensitized isologous lymph-node cells on the proliferation of C3H mammary cancer was studied. For this purpose tissue culture was conducted with regional lymph-node cells obtained from truly isologous C3H mouse inoculated with A strain cells derived from C3H mouse mammary cancer along with A cells, and the following results were obtained. In the case of tissue culture with those lymph-node cells obtained from the groups of mice 10 days after the inoculation of 5 X 106 A cells, the inhibitory effect on the proliferation of A cells was most marked, followed by that of those taken on day 14, 7, and 5 decreasing in the order mentioned. In the case with those regional lymph-node cells obtained from mice which did not have recurrence of tumor 1 week after extirpation of 2-week old tumor, the inhibitory effect on proliferation of A cells was marked, with the regional lymph-node cells obtained two weeks after transplantation of 1 × 108 A cells there could be observed no inhibitory effect at all. This suggests that at a certain stage after implantation of such regional lymph- node cells there develops a specific anti-tumor activity in the host.

Ultrastructure of microfilaria Brugia malayi was investigated with electron microscope. Microfilariae are covered by a sheath membrane with dense materials on its outer surfaces. The cuticle consists of 3 layers; namely, external cortical, internal cortical and fibrous layer. Beneath these cuticular layers, thin hypodermis is present and the muscle cells are arranged of 4 groups in a crosssection except for the head and tail. A pair of cephalic channel containing several cilial rods opens at the anterior end of the worm. A hook is situated on the anterior edge of one channel orifice, and several spines grow on the opposite side to the hook. Caudal channels paired laterally opening into the both sides of the posterior region differ from cephalic channels by the presence of a single cilial rod. A central canal runs from the buccal cavity to the inner body, and opens into the inner body cell through the filamentous apparatus. The inner body appears to consist of several cells having storage substances and a flat nucleus located on the periphery of the cell. An excretory apparatus, i. e., a cell, is composed of a nucleus and a large vesicle which has many microprojections on the luminal surfaces. The GI cell which occupies the whole width in a cross-section is larger than the R cell. R2-R4 cells appear to be in a close contact with the anal apparatus having many microprojections on the luminal surfaces. These microprojections differ from those of the excretory vesicle in their thickness and length. The characteristic patterns of these organs are compared with other microfilariae.

The mode of lymph flow from the breast to the regional lymph node was studied using 1llSAu colloid, to ascertain the concept of lymph node of Groups 1, 2, and 3. Lymph flow through the internal mammary nodes was found to be abundant. Especially so when 1llSAu colloid was injected into the lower half of the breast, there was observed more abundant flow than that to axillary lymph nodes. Therefore, it was concluded that regardless of the site of occupation of a tumor, internal mammary nodes should be con· sidered to be of Group 1. Lymph flow through the subclavian nodes proved to be always less than that to the axillary nodes or internal mammary nodes. The rate of the lymph flow appearance on the scintigram was about 20%. Whether the subclavian nodes should be classified as Group 1 or 2 should be further studied. The rate of supraclavicular node visualization was below 3%. Therefore, the supraclavicular nodes should be considered to be Group 2. When these results are applied to the current rule of handling of breast cancer, discrepancy occurs in the Stage number and R-numer.

Since detection of hepatitis C virus RNA by the polymerase chain reaction (PCR) showed that there existed anti-C100-3 (anti-HCV) antibody negative patients infected with HCV, we attempted to find out whether there were any clinical or viral genomic differences between the anti-HCV antibody positive and negative groups. One hundred and fifty-nine patients with chronic liver diseases with hepatitis C virus RNA in their sera were selected. Anti-HCV antibody was tested for anti-C100-3 antibody by an enzyme linked immunosorbent assay. The incidence of anti-HCV antibody was 129/159. The concentration of serum gamma-globulin, the titier of ZTT, and the positive rate of the PCR with the primers of the NS3/4 region (NS3/4PCR) were significantly higher in the anti-HCV antibody positive group than in the negative group. However, the other data such as alanine aminotransferase activity or past history were not significantly different. Nucleotide sequence of the cDNA fragments of NS3/4 region amplified by the PCR did not differ significantly between isolates from anti-HCV antibody positive and negative sera. The sequences observed in the present study did not differ significantly from those reported previously. Although there remains the possibility that the variation of viral genomic sequences may cause the absence of anti-HCV antibody, these results suggested that the individual clinical backgrounds or immunoreactivity of the patients might influence the antibody development.

We evaluated the surgical problems encountered during treatment of 14 patients with malignant tumors originating in the pelvic region at our department. The tumor involved the iliac bone in 6 patients, the ischial bone in 2, the pubic bone in 2, and the gluteal region in 4. Invasion to the sacrum was observed in 7 patients. Twelve patients underwent surgical procedures consisting of intralesional resection in 6, marginal resection in 3, and wide margin resection in 3. Six of the 7 patients with sacral invasion developed local recurrence. Two patients with chondrosarcoma and one with parosteal osteosarcoma survived for 4 or more years, but the mean survival period in those with high grade malignant tumors was 11 months. These findings indicate the difficulties encountered in the treatment of malignant pelvic tumors.

The amygdalofugal fibers were studied III the cat with the silver method of NAUTA-GYGAX. 1. The amygdalofugal fibers are distributed by way of the stria terminalis, the longitudinal association bundle, the inferior thalamic peduncle, and the medial forebrain bundle. 2. The amygdalofugal fibers running through the longitudinal association bundle arise in the lateral principal, intermediate principal nuclei and the lateral and possibly intermediate parts of the periamygdaloid cortex, and terminate in the lateral preoptic nucleus, the bed nucleus of the anterior commissure, the olfactory tubercle, the nucleus of the diagonal band of Broca, the nucleus accumbens, the medial and posterior septal nuclei and the basal part of the head of the caudate nucleus. In addition, there are scattered fibers coursing along the longitudinal association bundle proper. These fibers may have a widespread origin from the amygdaloid complex. The longitudinal association bundle contributes no fibers to the medial forebrain bundle. 3. The fibers, originating from the lateral principal, intermediate principal and medial principal nuclei, join the medial forebrain bundle to distribute widely in the lateral hypothalamic nucleus. A few fibers are seen to reach the ventromedial hypothalamic nucleus, and are considered to arise in the medial principal nucleus. 4. By way of the inferior thalamic peduncle some fibers from the amygdaloid complex course dorsally into the medial part of the dorsomedial thalamic nucleus at its caudal levels. They may arise widely from the amygdaloid complex. A few of them extend farther dorsally to reach the lateral habenular nucleus and the parataenial nucleus. They probably originate from the lateral principal nucleus. 5. The fibers forming the stria terminalis originate from the medial principal nucleus, the medial nucleus, the periamygdaloid cortex and the cortical nucleus, and are distributed in the bed nucleus of the stria terminalis and the lateral preoptic nucleus (preoptic component), as well as the medial preoptic nucleus, the anterior hypothalamic nucleus and the ventromedial hypothalamic nucleus (supracommissural component). The cortical nucleus, particularly its caudal part, and possibly the medial part of the periamygdaloid cortex are regarded as the main sources of the stria terminalis fibers ending in the hypothalamic region. The intermediate principal and lateral principal nuclei do not appear to contribute fibers to the stria terminalis. 6. The ventromedial hypothalamic nucleus receives amygdalofugal fibers both from the medial principal nucleus by way of the medial forebrain bundle, and from the cortical nucleus via the stria terminalis. 7. In addition to intrinsic internuclear fibers within the amygdaloid complex, some of the fibers from the complex are distributed to the ventralmost part of the putamen, the medial part of the claustrum, the periamygdaloid cortex, the prepiriform area and the anterior amygdaloid area, but do not reach the hippocampus.